ipsogen npm1 mutascreenrcostoya.com/uploads/galerias/con812/ipsogen-jak2-muta...july 2016 sample...

July 2016

Sample & Assay Technologies

ipsogen® NPM1 MutaScreen Handbook

24

For detection of NPM1 mutations and identification of mutation types A, B, and D

For research use only. Not for use in diagnostic procedures.

For use with Rotor-Gene® Q, Applied Biosystems® 7500 Real-Time PCR System, ABI PRISM® 7900HT SDS, and LightCycler® 480 instruments

677013

QIAGEN GmbH, QIAGEN Strasse 1, 40724 Hilden, GERMANY

R3

QIAGEN Sample and Assay Technologies QIAGEN is the leading provider of innovative sample and assay technologies, enabling the isolation and detection of contents of any biological sample. Our advanced, high-quality products and services ensure success from sample to result.

QIAGEN sets standards in:

Purification of DNA, RNA, and proteins

Nucleic acid and protein assays

microRNA research and RNAi

Automation of sample and assay technologies

Our mission is to enable you to achieve outstanding success and breakthroughs. For more information, visit www.qiagen.com.

ipsogen NPM1 MutaScreen Handbook 07/2016 3

Contents Intended Use 4

Principle of the Procedure 4

Materials Provided 6

Kit contents 6

Materials Required but Not Provided 6

Warnings and Precautions 7

General precautions 7

Reagent Storage and Handling 8

Procedure 9

Sample DNA preparation 9

Protocols

qPCR on Rotor-Gene Q 5plex HRM instruments with 72-tube rotor 10

qPCR on Applied Biosystems 7500 and ABI PRISM 7900HT instruments, and LightCycler 480 instrument 14

Results 19

Step by step interpretation of results 21

Troubleshooting guide 25

Quality Control 28

Symbols 28

Contact Information 28

Ordering Information 29

4 ipsogen NPM1 MutaScreen Handbook 07/2016

Intended Use The ipsogen NPM1 MutaScreen Kit is intended for research use only. Not for use in diagnostic procedures. No claim or representation is intended to provide information for the diagnosis, prevention, or treatment of a disease.

All due care and attention should be exercised in the handling of the products. We recommend all users of QIAGEN® products to adhere to the NIH guidelines that have been developed for recombinant DNA experiments, or to other applicable guidelines.

Principle of the Procedure The ipsogen NPM1 MutaScreen Kit combines two techniques to screen for the presence of mutations in the target gene. The real-time quantitative PCR (qPCR) double-dye oligonucleotide hydrolysis principle uses specific primers and an internal double-dye probe with a reporter and a quencher (FAM™-TAMRA™) for the amplification reactions. In addition, a 3'-end modified phosphate oligonucleotide is used that perfectly matches the wild-type NPM1 gene and does not allow polymerization.

During PCR, if the target of interest is present, the probe specifically anneals between the forward and reverse primer sites. The DNA polymerase cleaves the probe between the reporter and the quencher only if the probe hybridizes to the target. The probe fragments are then displaced from the target, polymerization of the strand continues, and a fluorescent signal is released. The 3' end of the probe is blocked to prevent extension of the probe during PCR (see Figure 1).

The phosphate oligonucleotide enhances specificity of the PCR reaction by competition with one PCR primer for a common target site. When the PCR template contains the wild-type sequence, the phosphate oligonucleotide will dominate over PCR primer binding due to higher affinity. There is no extension by the DNA polymerase, and no amplification is observed. When the mutated sequence is present, PCR primer binding will dominate over phosphate oligonucleotide binding and amplification will proceed.

The ipsogen NPM1 MutaScreen Kit detects, total NPM1 (wild-type + mutated), mutated NPM1 and separately identifies NPM1 Mut A, Mut B and Mut D in genomic DNA. The ipsogen NPM1 MutaScreen Kit provides two results simultaneously: NPM1 mutation status (mutated or wild-type) and identification of NPM1 mutation type (A, B or D).


Figure 1. Results obtained with the primers and probe mixes in the ipsogen NPM1 MutaScreen Kit. The same principle shown to detect NPM1 MutA applies for NPM1 MutB and NPM1 MutD.


Materials Provided Kit contents

ipsogen NPM1 MutaScreen Kit (24)

Catalog no. 677013

Number of reactions 24

NPM1 wild-type control (wild-type NPM1 genomic DNA)

WTC 300 µl

NPM1 mutated positive control (NPM1 MutA/B/D genomic DNA)

Mut-PC 300 µl

Primers and Probe Mix Total NPM1* PPM-Total NPM1 25x 90 µl

Primers and Probe Mix Mutated NPM1† PPM-Mut NPM1 25x 90 µl

Primers and Probe Mix NPM1 MutA‡ PPM-NPM1 MutA 25x 90 µl

Primers and Probe Mix NPM1 MutB§ PPM-NPM1 MutB 25x 90 µl

Primers and Probe Mix NPM1 MutD¶ PPM-NPM1 MutD 25x 90 µl

* Mix of specific reverse and forward primers for total NPM1 (wild-type and mutated) plus a specific FAM–TAMRA probe.

† Mix of specific reverse and forward primers (including phosphate primer) for all mutations of NPM1 plus a specific FAM–TAMRA probe.

‡ Mix of specific reverse and forward primers (including phosphate oligonucleotide) for the specific detection of NPM1 MutA plus a specific FAM–TAMRA probe.

§ Mix of specific reverse and forward primers (including phosphate oligonucleotide) for the specific detection of NPM1 MutB plus a specific FAM–TAMRA probe.

¶ Mix of specific reverse and forward primers (including phosphate oligonucleotide) for the specific detection of NPM1 MutD plus a specific FAM–TAMRA probe.

Note: Vortex and briefly centrifuge the controls and the primers and probe mixes before use.

Materials Required but Not Provided When working with chemicals, always wear a suitable lab coat, disposable gloves, and protective goggles. For more information, consult the appropriate safety data sheets (SDSs), available from the product supplier.


Reagents

Nuclease-free PCR-grade water

Buffer and Taq DNA polymerase: The recommended reagents are TaqMan® Universal PCR Master Mix (Master Mix PCR 2x) (Thermo Fisher Scientific Inc., cat. no. 4304437)

Consumables

Nuclease-free aerosol-resistant sterile PCR pipet tips with hydrophobic filters

0.5 ml or 1.5 ml nuclease-free PCR tubes

Ice

Equipment

Microliter pipet*dedicated for PCR (1–10 µl; 10–100 µl; 100–1000 µl)

Benchtop centrifuge* with rotor for 0.5 ml/1.5 ml reaction tubes and microplates (capable of attaining 13,000–14,000 rpm)

Real-time PCR instrument:* Rotor-Gene Q 5plex HRM or other Rotor-Gene instrument; LightCycler 480; Applied Biosystems 7500 Real-Time PCR System; ABI PRISM 7900HT SDS; and associated specific material

Spectrophotometer*

Warnings and Precautions When working with chemicals, always wear a suitable laboratory coat, disposable gloves, and protective goggles. For more information, please consult the appropriate safety data sheets (SDSs). These are available online in convenient and compact PDF format at www.qiagen.com/safety where you can find, view, and print the SDS for each QIAGEN kit and kit component.

Discard sample and assay waste according to your local safety regulations.

General precautions Use of qPCR tests require good laboratory practices, including maintenance of equipment, that are dedicated to molecular biology and is compliant with applicable regulations and relevant standards.

* Ensure that instruments have been checked and calibrated according to the manufacturer’s

recommendations.


This kit is intended for research use. Reagents and instructions supplied in this kit have been tested for optimal performance. Further dilution of the reagents or alteration of incubation times and temperatures may result in erroneous or discordant data. Primers and probe mix (PPM) reagents may be altered if exposed to light. All reagents are formulated specifically for use with this kit. For optimal performance of the kit, no substitutions should be made.

Use extreme caution to prevent:

DNase contamination that might cause degradation of the template DNA

DNA or PCR carryover contamination resulting in false positive signal

We therefore recommend the following.

Use nuclease-free labware (e.g., pipets, pipet tips, reaction vials) and wear gloves when performing the assay.

Use fresh aerosol-resistant pipet tips for all pipetting steps to avoid cross-contamination of the samples and reagents.

Prepare pre-PCR master mix with dedicated material (pipets, tips, etc.) in a dedicated area where no DNA matrices (DNA, plasmid, or PCR products) are introduced. Add template in a separate zone (preferably in a separate room) with specific material (pipets, tips, etc.).

Reagent Storage and Handling The kits are shipped on dry ice and must be stored at –30°C to –15°C upon receipt.

Minimize exposure to light of the primers and probe mixes (PPM tubes).

Gently mix and centrifuge the tubes before opening.

Store all kit components in original containers.

These storage conditions apply to both opened and unopened components. Components stored under conditions other than those stated on the labels may not perform properly and may adversely affect the assay results.

Expiration dates for each reagent are indicated on the individual component labels. Under correct storage conditions, the product will maintain performance until the expiration date printed on the label.

There are no obvious signs to indicate instability of this product. However, positive and negative controls should be run simultaneously with unknown specimens.


Procedure

Sample DNA preparation Genomic DNA should be obtained either from whole blood, purified peripheral blood lymphocytes of whole blood, polynuclear cells, or granulocytes. For comparable results it is recommended that the same cellular fraction and DNA extraction method are used. DNA extraction can be performed using a commercially available kit or a home-brew method.

DNA quantity should be determined by measuring the optical density (OD) of the sample at 260 nm and DNA quality can be determined either by spectrophotometry or gel* electrophoresis.

The OD260/OD280 ratio should be 1.7–1.9 and smaller ratios than this may indicate protein contamination or the presence of organic chemicals.

Electrophoretic analysis on a 0.8–1.0% agarose gel* should allow the visualization of the isolated DNA as a distinct band of approximately 20 kb (a slight smear will give acceptable results).

The resultant DNA will need to be diluted to a concentration of 5 ng/µl in 1x TE buffer* at pH 8.0 and then stored at +4 to +8°C for 1 week or at –20°C if longer term storage is required.

The qPCR reaction is optimized for DNA samples containing 25 ng purified genomic DNA. An amount of 250 ng of genomic DNA is needed per sample.

* When working with chemicals, always wear a suitable lab coat, disposable gloves, and

protective goggles. For more information, consult the appropriate safety data sheets (SDSs), available from the product supplier.


Protocol: qPCR on Rotor-Gene Q 5plex HRM instruments with 72-tube rotor Using this instrument, we recommend performing all measurements in duplicate, as indicated in Table 1.

Table 1. Number of reactions for Rotor-Gene Q instruments with 72-tube rotor

Samples Reactions

With the total NPM1 primers and probe mix (PPM-Total NPM1)

With the mutated NPM1 primers and probe mix (PPM-Mut NPM1)

With the NPM1 MutA primers and probe mix (PPM-NPM1 MutA)

With the NPM1 MutB primers and probe mix (PPM-NPM1 MutB)

With the NPM1 MutD primers and probe mix (PPM-NPM1 MutD)

n DNA samples n x 2 reactions

2 DNA controls

4 reactions: wild-type control (WTC) and mutated positive control (Mut-PC), each one tested in duplicate

Water control 2 reactions

Sample processing on Rotor-Gene Q instruments with 72-tube rotor

We recommend testing four DNA samples in the same experiment, 14 reactions with each primers and probe mix, to optimize the use of controls and the primers and probe mixes.


Figure 3. Suggested rotor setup for an experiment with the ipsogen NPM1 MutaScreen Kit. WTC: wild-type NPM1positive control; Mut-PC: mutated NPM1 positive control; S: DNA sample; H2O: water control.

Note: Take care to always place a sample to be tested in position 1 of the rotor. Otherwise, during the calibration step, the instrument will not perform calibration, and incorrect fluorescence data will be acquired.

Fill all other positions with empty tubes.

qPCR on Rotor-Gene Q instruments with 72-tube rotor

Note: Perform all steps on ice.

Procedure

1. Thaw all necessary components and place them on ice. 2. Prepare the following qPCR mix for each primers and probe mix

according to the number of samples being processed. All concentrations are for the final volume of the reaction.


Table 2 describes the pipetting scheme for the preparation of one reagent mix, calculated to achieve a final reaction volume of 25 µl. A pre-mix can be prepared, according to the number of reactions, using the same primer and probe mix (PPM-Total NPM1, PPM-Mut NPM1, PPM-NPM1 MutA, PPM-NPM1 MutB, or PPM-NPM1 MutD). Extra volumes are included to compensate for pipetting error.

Table 2. Preparation of qPCR mix for PPM-Total NPM1, PPM-Mut NPM1, PPM-NPM1 MutA, PPM-NPM1 MutB, or PPM-NPM1 MutD

Component 1 reaction

(µl)

Pre-mix 14 + 1

reactions (µl) Final

concentration

TaqMan Universal PCR Master Mix, 2x

12.5 187.5 1x

Primers and probe mix, PPM 25x

1.0 15 1x


6.5 97.5 –

Sample (to be added at step 4)

5.0 5 each –

Total volume 25.0 25 each –

3. Dispense 20 µl of the qPCR pre-mix per tube. 4. Add 5 µl of the material to be quantified (25 ng sample genomic

DNA or control) in the corresponding tube (total volume 25 µl). 5. Mix gently, by pipetting up and down. 6. Place the tubes in the thermal cycler according to the manufacturer

recommendations. 7. Program the Rotor-Gene Q instrument with the thermal cycling

program as indicated in Table 3.


Table 3. Temperature profile

Mode of analysis Quantitation

Hold Temperature: 50 deg

Time: 2 mins

Hold 2 Temperature: 95 deg

Time: 10 mins

Cycling 40 times

95 deg for 15 secs

60 deg for 1 min 30 secs with acquisition of FAM fluorescence in channel Green: Single

8. For Rotor-Gene Q instruments, select “Slope Correct” for the analysis. We recommend setting the threshold at 0.03. Start the thermal cycling program, as indicated in Table 3.


Protocol: qPCR on Applied Biosystems 7500 and ABI PRISM 7900HT instruments, and LightCycler 480 instrument Using 96-well-plate qPCR equipment, we recommend performing all measurements in duplicate, as indicated in Table 4.

Table 4. Number of reactions using 96-well plate qPCR equipment

Samples Reactions

With the total NPM1 primers and probe mix (PPM-Total NPM1)

With the mutated NPM1 primers and probe mix (PPM-Mut NPM1)

With the NPM1 MutA primers and probe mix (PPM-NPM1 MutA)

With the NPM1 MutB primers and probe mix (PPM-NPM1 MutB)

With the NPM1 MutD primers and probe mix (PPM-NPM1 MutD)

n DNA samples n x 2 reactions

2 DNA controls

4 reactions: wild-type control (WTC) and mutated positive control (Mut-PC), each one tested in duplicate

Water control 2 reactions

Sample processing on Applied Biosystems 7500 and ABI PRISM 7900HT instruments, and LightCycler 480 instrument

We recommend testing 4 DNA samples in the same experiment, 14 reactions with each primers and probe mix, to optimize the use of controls and the primers and probe mixes.


Figure 4. Suggested plate setup for an experiment with the ipsogen NPM1 MutaScreen Kit. WTC: wild-type NPM1positive control; Mut-PC: mutated NPM1 positive control; S: DNA sample; H2O: water control. S: DNA sample; H2O: water control

qPCR on Applied Biosystems 7500 and ABI PRISM 7900HT instruments, and LightCycler 480 instrument

Note: Perform all steps on ice.

Procedure

1. Thaw all necessary components and place them on ice. 2. Prepare the following qPCR mix for each primers and probe mix

according to the number of samples being processed. All concentrations are for the final volume of the reaction.

Table 5 describes the pipetting scheme for the preparation of one reagent mix, calculated to achieve a final reaction volume of 25 µl. A pre-mix can be prepared, according to the number of reactions, using the same primer and probe mix (PPM-Total NPM1, PPM-Mut NPM1, PPM-NPM1 MutA, PPM-NPM1 MutB, or PPM-NPM1 MutD). Extra volumes are included to compensate for pipetting error.


Table 5. Preparation of qPCR mix for PPM-Total NPM1, PPM-Mut NPM1, PPM-NPM1 MutA, PPM-NPM1 MutB, or PPM-NPM1 MutD

Component 1 reaction

(µl)

Pre-mix 14 + 1

reactions (µl) Final

concentration

TaqMan Universal PCR Master Mix, 2x

12.5 187.5 1x

Primers and probe mix, PPM 25x

1.0 15 1x


6.5 97.5 –

Sample (to be added at step 4)

5.0 5 each –

Total volume 25.0 25 each –

3. Dispense 20 µl of the qPCR pre-mix per well. 4. Add 5 µl of the material to be quantified (25 ng sample genomic

DNA or control) in the corresponding well (total volume 25 µl). 5. Mix gently, by pipetting up and down. 6. Close the plate and briefly centrifuge (300 x g, approximately 10 s). 7. Place the plate in the thermal cycler according to the manufacturer

recommendations. 8. Program the thermal cycler with the thermal cycling program and set

the instrument for the acquisition of dual labeled FAM fluorescent probe as indicated in Table 6 for Applied Biosystems 7500 and ABI PRISM 7900HT instruments, or Table 7 for the LightCycler 480 instrument.


Table 6. Temperature profile for Applied Biosystems 7500, ABI PRISM 7000, ABI PRISM 7700, or ABI PRISM 7900HT instruments

Mode of analysis Standard Curve — Absolute Quantitation

Hold Temperature: 50°C

Time: 2 minutes

Hold 2 Temperature: 95°C

Time: 10 minutes

Cycling 40 times

95°C for 15 seconds

60°C for 1 minute 30 seconds with acquisition of FAM fluorescence; quencher: TAMRA

Table 7. Temperature profile for LightCycler 480 instrument

Mode of analysis Absolute Quantification (“Abs Quant”)

Detection formats Select “Simple Probe” in the Detection formats window

Hold Temperature: 50°C

Time: 2 minutes

Hold 2 Temperature: 95°C

Time: 10 minutes

Cycling 40 times

95°C for 15 seconds

60°C for 1 minute 30 seconds with acquisition of FAM fluorescence corresponding to (483–533 nm) for LC version 01 and (465–510 nm) for LC version 02


9. For the Applied Biosystems 7500 and ABI PRISM 7900HT instruments, follow step 9a. For the LightCycler 480 instrument, follow step 9b.

9a. Applied Biosystems 7500 and ABI PRISM 7900HT instruments: We recommend a threshold set at 0.1 and a baseline set between cycles 3 and 15. Start the cycling program, as indicated in Table 6.

9b. LightCycler 480: We recommend a Fit point analysis mode with background at 2.0 and threshold at 2.0. Start the thermal cycling program, as indicated in Table 7.


Results Calculate the mean threshold cycle (CT) value obtained with each primers and probe mix for the controls (NPM1 wild-type control and mutated NPM1 positive control) and for each sample. If one of the duplicates of a sample has an “undetermined” value, we recommend retesting the sample.

Quality control using CT values of controls

The NPM1 wild-type control (WTC) and the mutated NPM1 positive control (Mut-PC) allow an experiment to be qualified.

The NPM1 wild-type control must be detected with only PPM-Total NPM1.

The mutated NPM1 positive control must be detected with all PPMs.

The entire experiment is rejected if both conditions are not met.

Water controls

Water controls (non-template controls) should give zero CT values for all primers and probe mixes.

A positive water control results from a cross-contamination. See “Troubleshooting guide”, page 25, to find a solution.

Sample input validation

A sample’s input must be validated before interpretation. The validation interval is determined with these calculations.

CT Total NPM1 (WTC): CT value of the NPM1 wild-type control with PPM-Total NPM1

CT Total NPM1 (Mut-PC): CT value of the mutated NPM1 positive control with PPM-Total NPM1

CT Total NPM1 (Sample): CT value of a sample with PPM-Total NPM1

The value of CT Total NPM1 (Sample) must be within the following interval:

[CT Total NPM1 (WTC); CT Total NPM1 (Mut-PC) + 2.3]

The sample is positive for an NPM1 mutation if a CT value is obtained with the primers and probe mix PPM-Mut NPM1. This is CT Mut NPM1 (Sample).


The NPM1 mutation type of a sample, mutA, MutB, or MutD, is determined by the CT values obtained with PPM-NPM1 MutA, PPM-NPM1 MutB, and PPM-NPM1 MutD. The mutation type is indicated by the result with the lowest CT value, providing that CT value respects the following rule:

CT NPM1 MutA, MutB, or MutD (Sample) ≤ CT Mut NPM1 (Sample) + 4.4

A summary of the rules of sample validation and analysis is given in Table 8.

Table 8. Successive rules applied for run validation and results analysis

No. Stage Rule

1

Quality control

Water controls

Must give zero CT values

2 NPM1 wild-type control

Must be detected with only PPM-total NPM1

3

Mutated NPM1 positive control

Must be detected with all primer and probe mixes

4 Sample input

Input interval

[CT Total NPM1 (WTC);CT Total NPM1 (Mut-PC)+2.3]

5 Sample validation

The value of CT Total NPM1 (Sample) must be within the input interval

6

Analysis

Mutation detection

A sample is positive for an NPM1 mutation if a CT value is obtained with PPM-Mut NPM1: CT Mut NPM1 (Sample)

7 Mutation type detection

The NPM1 mutation type of a sample is determined by the lowest CT value obtained with PPM-NPM1 MutA, PPM-NPM1 MutB, and PPM-NPM1 MutD providing CT NPM1 MutA, MutB, or MutD (Sample) ≤CT Mut NPM1 (Sample) + 4.4


Step by step interpretation of results Interpretation is shown of results from 9 samples plus controls analyzed with the ipsogen NPM1 MutaScreen Kit.

Quality control

Quality control using CT values of controls is shown in Table 9.

Table 9. Run validation on controls

Mean CT values with primers and probe mixes

Control Total NPM1

Mut NPM1

NPM1 MutA

NPM1 MutB

NPM1 MutD

Water (negative)

Undetected Undetected Undetected Undetected Undetected

NPM1 WTC

23.84 Undetected Undetected Undetected Undetected

NPM1 Mut-PC

26.9 30.49 31.88 32.82 33.09

Quality control shows that the run is valid. Conditions of rules 1, 2, and 3 have been met:

Rule 1: Water control is undetected with all primer and probe mixes.

Rule 2: NPM1 WTC is detected with only PPM-Total NPM1.

Rule 3: NPM1-Mut PC is detected with all primer and probe mixes.

Input interval

Determination of the input interval using CT values of positive controls is shown.

Input interval [CT Total NPM1 (WTC); CT Total NPM1 (Mut-PC) + 2.3]

[23.84; 26.9 + 2.3]

Input interval [23.84; 29.2]

Rule 4: The input interval for sample validation is determined.


Sample validation

Sample input validation using the input interval is shown in Table 10.

Table 10. Sample input validation

Mean CT values with primers and probe mixes

Sample Total NPM1

Mut NPM1

NPM1 MutA

NPM1 MutB

NPM1 MutD

1 26.46 27.66 28.29 Undetected 38.37

2 26.12 32.73 32.78 Undetected Undetected

3 27.68 29.12 Undetected 29.79 Undetected

4 28.78 34.92 Undetected 33.57 Undetected

5 26.36 27.49 36.28 Undetected 27.96

6 26.56 33.73 Undetected Undetected 33.54

7 25.81 27.06 37.23 38.7 Undetected

8 26.89 27.87 38.91 Undetected Undetected

9 25.64 Undetected Undetected Undetected Undetected

Results of the samples are valid. Conditions of Rule 5 have been met.

Rule 5: The values of the CT Total NPM1 (Sample) for all samples are within the input interval [23.84; 29.2].

NPM1 mutation detection

Detection of an NPM1 mutation in the samples is shown in Table 11.


Table 11. NPM1 mutation detection

Mean CT values with PPM Screening

Sample Total NPM1

Mut NPM1

Mutation Detected Type

1 26.46 27.66 Yes MutA

2 26.12 32.73 Yes MutA

3 27.68 29.12 Yes MutB

4 28.78 34.92 Yes MutB

5 26.36 27.49 Yes MutD

6 26.56 33.73 Yes MutD

7 25.81 27.06 Yes Not typed

8 26.89 27.87 Yes Not typed

9 25.64 Undetected No NA

Samples positive for NPM1 mutation are detected: CT Mut NPM1 (Sample). Conditions of Rule 6 have been met for 8 of 9 samples.

Rule 6: A sample is positive for an NPM1 mutation if a CT value is obtained with PPM-Mut NPM1.

NPM1 mutation type

The procedure used to assign the NPM1 mutation type to samples positive for an NPM1 mutation is shown in Table 12.


Table 12. Typing of NPM1 mutation as MutA, MutB, or MutD

Mean CT values with PPM

Sample Mut NPM1

NPM1 MutA

NPM1 Mut B

NPM1 MutD Type

1 27.66 28.29 Undetected 38.37 MutA

2 32.73 32.78 Undetected Undetected MutA

3 29.12 Undetected 29.79 Undetected MutB

4 34.92 Undetected 33.57 Undetected MutB

5 27.49 36.28 Undetected 27.96 MutD

6 33.73 Undetected Undetected 33.54 MutD

7 27.06 37.23 38.7 Undetected Not typed

8 27.87 38.91 Undetected Undetected Not typed

9 Undetected Undetected Undetected Undetected NA

If a sample has CT values for more than one of the primers and probe mixes for detecting MutA, MutB, or MutD mutations, the lowest of these CT values for the sample is used to assign the mutation type. See sample 1 and sample 5 in Table 12. Samples may be genotyped providing the conditions of Rule 7 are met. Conditions of Rule 7 have been met in 6 of 8 samples.

Rule 7: The NPM1 mutation type of a sample is determined by the lowest CT value obtained with PPM-NPM1 MutA, PPM-NPM1 MutB, and PPM-NPM1 MutD providing CT NPM1 MutA, MutB, or MutD (Sample) ≤ CT Mut NPM1 (Sample) + 4.4.

For sample 7 and sample 8, the CT NPM1 MutA (Sample) values (37.23 and 38.91) are greater than the CT Mut NPM1 (Sample) values + 4.4. These values are 31.46 and 32.27 respectively.


Troubleshooting guide This troubleshooting guide may be helpful in solving any problems that may arise. For more information, see also the Frequently Asked Questions page at our Technical Support Center: www.qiagen.com/FAQ/FAQList.aspx. The scientists in QIAGEN Technical Services are always happy to answer any questions you may have about either the information and protocol in this handbook or sample and assay technologies (for contact information, see “Contact Information”, page 28).

Comments and suggestions

NPM1 wild-type control not detected with PPM-Total NPM1

a) Pipetting errors or omitted reagents; tube or well inversions

Check pipetting scheme and the setup of the reaction.

Repeat the PCR run.

b) Inappropriate storage of kit components

Aliquot reagents for storage.

Store the ipsogen NPM1 MutaScreen Kit at –15 to –30°C and keep primers and probe mixes protected from light. See “Reagent Storage and Handling”, page 8.

Avoid repeated freezing and thawing.

NPM1 mutated positive control not detected with PPM-Mut NPM1, PPM-NPM1 MutA, PPM-NPM1 MutB, or PPM-NPM1 MutD

a) Pipetting errors or omitted reagents; tube or well inversions


Repeat the PCR run.

b) Inappropriate storage of kit components




No signal, even in controls

a) In Rotor-Gene Q instruments, no reaction tube in position 1

Take care to always place a sample to be tested in position 1 of the rotor.



b) Pipetting errors or omitted reagents


Repeat the PCR run.

c) Inappropriate storage of kit components




d) Incorrect detection channel has been chosen

Set channel to Green channel or 530 nm/640 nm.

e) No data acquisition program Check the cycle program.

Select acquisition mode “Single” at the end of each annealing segment of the PCR program.

Fluorescence intensity varies

a) Pipetting errors Check pipetting scheme and the setup of the reaction.

Vortex and spin all reagents after thawing.

Repeat the PCR run.

b) Insufficient centrifugation of the tubes or plate

Always centrifuge tubes or plates loaded with the reaction mix as described in the specific operating manual of the apparatus.

Inconsistent CT values between duplicates

a) Pipetting errors or cross-contamination


Repeat the PCR run.

b) Low mutation load Always check the DNA quality (OD260/OD280) and concentration before starting.



Fluorescence intensity is too low

a) Inappropriate storage of kit components




b) Very low initial amount of target DNA

Check the amount of sample DNA.

Always check the DNA quality (OD260/OD280) and concentration before starting.

Note: Depending of the chosen method of DNA preparation, inhibitory effects may occur.

Signal absent or low in samples but okay in controls

Inhibitory effects of sample material caused by insufficient purification

Always check the DNA quality (OD260/OD280) and concentration before starting.

Repeat DNA preparation.

Negative (H2O) control is positive

Cross-contamination, reagent contamination, instrument error, well or capillary inversion, or probe degradation

Replace all critical reagents.

Always handle samples, kit components, and consumables in accordance with commonly accepted practices to prevent carry over contamination.

Keep primers and probe mixes protected from light.

Check for false positives on fluorescence curves.

Check the setup of the reaction.


Quality Control In accordance with QIAGEN’s ISO-certified Quality Management System, each lot ipsogen NPM1 MutaScreen Kit is tested against predetermined specifications to ensure consistent product quality. Certificates of analysis are available on request at www.qiagen.com/support/.

Symbols The following symbols may appear on the packaging and labeling:

<N> Contains reagents sufficient for <N> reactions

Use by

Catalog number

Lot number

Material number

Global Trade Item Number

Temperature limitation

Manufacturer

Consult instructions for use

Contact Information For technical assistance and more information, please see our Technical Support Center at www.qiagen.com/Support, call 00800-22-44-6000, or contact one of the QIAGEN Technical Service Departments or local distributors (see back cover or visit www.qiagen.com).


Ordering Information Product Contents Cat. no.

ipsogen NPM1 MutaScreen Kit (24)

For 24 reactions: Wild-type NPM1 Control, Mutated NPM1 Control , Primers and Probe Mix PPM-Total NPM1, Primers and Probe Mix PPM-Mutated NPM1, Primers and Probe Mix PPM-NPM1 MutA, Primers and Probe Mix PPM-NPM1 MutB, Primers and Probe Mix PPM-NPM1 MutD

677013

Rotor-Gene Q – for outstanding performance in real-time PCR

Rotor-Gene Q 5plex HRM Platform

Real-time PCR cycler and High Resolution Melt analyzer with 5 channels (green, yellow, orange, red, crimson) plus HRM channel, laptop computer, software, accessories, 1-year warranty on parts and labor, installation and training not included

9001580

Rotor-Gene Q 5plex HRM System

Real-time PCR cycler and High Resolution Melt analyzer with 5 channels (green, yellow, orange, red, crimson) plus HRM channel, laptop computer, software, accessories, 1-year warranty on parts and labor, installation and training

9001650

For up-to-date licensing information and product-specific disclaimers, see the respective QIAGEN kit handbook or user manual. QIAGEN kit handbooks and user manuals are available at www.qiagen.com or can be requested from QIAGEN Technical Services or your local distributor.


Notes

This product is intended to be used for life science research only. It is not intended for diagnostic use. ipsogen products may not be resold, modified for resale, or used to manufacture commercial products without written approval of QIAGEN. Information in this document is subject to change without notice. QIAGEN assumes no responsibility for any errors that may appear in this document. This document is believed to be complete and accurate at the time of publication. In no event shall QIAGEN be liable for incidental, special, multiple, or consequential damages in connection with, or arising from the use of this document.

ipsogen products are warranted to meet their stated specifications. QIAGEN’s sole obligation and the customer's sole remedy are limited to replacement of products free of charge in the event products fail to perform as warranted.

The purchase of this product allows the purchaser to use it for the performance of diagnostic services for human in vitro diagnostics. No general patent or other license of any kind other than this specific right of use from purchase is granted hereby.

NPM1 mutations and uses thereof are protected by patent rights, including European patent applications EP1807448, EP1944316 and EP2319865, US patent 8,222,370, US patent application US2008299560, and foreign counterparts.

Trademarks: QIAGEN®, Sample to Insight®, ipsogen®, Rotor-Gene® (QIAGEN Group); ABI PRISM®, Applied Biosystems®, FAM™, TAMRA™ (Thermo Fisher Scientific Inc.); LightCycler®, TaqMan® (Roche Group).

Limited License Agreement for ipsogen NPM1 MutaScreen Kit

Use of this product signifies the agreement of any purchaser or user of the product to the following terms:

1. The product may be used solely in accordance with the protocols provided with this product and this handbook and for use with components contained in the Kit only. QIAGEN grants no license under any of its intellectual property to use or incorporate the enclosed components of this Kit with any components not included within this Kit except as described in the protocols provided with this product, this handbook, and additional protocols available at www.qiagen.com. Some of these additional protocols have been provided by QIAGEN users for QIAGEN users. These protocols have not been thoroughly tested or optimized by QIAGEN. QIAGEN neither guarantees them nor warrants that they do not infringe the rights of third-parties.

2. Other than expressly stated licenses, QIAGEN makes no warranty that this Kit and/or its use(s) do not infringe the rights of third-parties.

3. This Kit and its components are licensed for one-time use and may not be reused, refurbished, or resold.

4. QIAGEN specifically disclaims any other licenses, expressed or implied other than those expressly stated.

5. The purchaser and user of the Kit agree not to take or permit anyone else to take any steps that could lead to or facilitate any acts prohibited above. QIAGEN may enforce the prohibitions of this Limited License Agreement in any Court, and shall recover all its investigative and Court costs, including attorney fees, in any action to enforce this Limited License Agreement or any of its intellectual property rights relating to the Kit and/or its components.

For updated license terms, see www.qiagen.com.

HB-1421-003

© 2016 QIAGEN, all rights reserved.

1100284 07/2016 Sample & Assay Technologies

www.qiagen.com

ipsogen npm1 mutascreenrcostoya.com/uploads/galerias/con812/ipsogen-jak2-muta...july 2016 sample...

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